EPH-like receptor protein tyrosine kinases

ABSTRACT

Four novel members of the EPH sub-family of receptor protein tyrosine kinases are disclosed. Nucleic acid sequences encoding receptor proteins, recombinant plasmids and host cells for expression, and methods of producing and using such receptors are also disclosed.

This application is a continuation of U.S. patent application Ser. No.09/378,759, filed Aug. 23, 1999; which is a divisional of U.S. patentapplication Ser. No. 08/702,367, filed Aug. 21, 1996, now U.S. Pat. No.5,981,246; which is a continuation of U.S. patent application Ser. No.08/229,509, filed Apr. 15, 1994, now abandoned, all of which areincorporated herein by reference for any purpose.

FIELD OF THE INVENTION

The invention relates generally to receptor protein tyrosine kinases(PTKs) and particularly to novel Eph-like receptor PTKs, to fragmentsand analogs thereof, and to nucleic acids encoding same. The presentinvention also relates to methods of producing and using such receptors.

BACKGROUND OF THE INVENTION

Receptor PTKs are a structurally related family of proteins that mediatethe response of cells to extracellular signals (Ullrich et al. Cell 61,203-212 (1990)). These receptors are characterized by three majorfunctional domains: an intracellular region containing the sequencesresponsible for catalytic activity, a single hydrophobicmembrane-spanning domain, and a glycosylated extracellular region whosestructure determines ligand binding specificity. Signal transduction isinitiated by the binding of growth or differentiation factors to theextracellular domain of their cognate receptors. Ligand bindingfacilitates dimerization of the receptor which can induce receptorautophosphorylation. Both soluble and membrane-associated proteinligands have been shown to function in this manner. This process is theinitial step in a cascade of interactions involving the phosphorylationof a variety of cytoplasmic substrates and culminating in a biologicalresponse by the cell. The best characterized response to tyrosine kinasereceptor activation is cell growth. However, analysis of the role ofsome growth factors in vivo suggests that differentiation or cellsurvival might also be mediated by tyrosine kinase receptor/ligandinteractions.

Receptor PTKs have been grouped into fairly well-defined families on thebasis of both sequence homology and shared structural motifs. The aminoacid sequence of the portion of the intracellular domain responsible forthe catalytic activity is well conserved among all tyrosine kinases andeven more closely matched within a receptor sub-family. Comparisons ofthis portion of the amino acid sequence have been used to constructphylogenetic trees depicting the relatedness of family members to eachother and to the tyrosine kinases as a whole (Hanks and Quinn, MethodsEnzymol. 200, 38-62 (1991)). This sequence conservation has also beenexploited in order to isolate new tyrosine kinases using the polymerasechain reaction (PCR) (Wilks, Proc. Natl. Acad. Sci. USA 86, 1603-1607(1989)). Oligonucleotides based on the highly conserved catalytic domainof PTKs can be used as PCR primers to amplify related sequences presentin the template. These fragments can then be used as probes forisolation of the corresponding full-length receptor clones from cDNAlibraries. Anti-phosphotyrosine antibodies have also been used toidentify PTK cDNA clones in phage expression libraries (Lindberg andPasquale, Methods Enzymol. 200, 557-564 (1991)). These strategies havebeen used by a number of investigators to identify an ever-increasingnumber of protein tyrosine kinase receptors.

There are now 51 distinct PTK receptor genes that have been publishedand divided into 14 sub-families One such sub-family is the EPH-likereceptors. The prototype member, EPH, was isolated by Hirai et. al.(Science 238, 1717-1720 (1987)) using low stringency hybridization to aprobe derived from the viral oncogene v-fps. EPH-like receptors havebeen implicated in cell growth based in part on studies which show thatoverexpression of the gene in NIH3T3 cells causes focus formation insoft agar and tumors in nude mice (Maru et al. Oncogene 5, 199-204(1990)). Other members of the EPH sub-family which have been identifiedinclude the following:

ECK (Lindberg et al. Mol. Cell. Biol. 10, 6316-6324 (1990))

Elk (Lhoták et al. Mol. Cell. Biol. 11, 2496-2502 (1991))

Ceks 4, 5, 6, 7, 8, 9, and 10 (Pasquale, Cell Regulation 2, 523-534(1991); Sajjadi et al. The New Biologist 3, 769-778 (1991); Sajjadi andPasquale Oncogene 8, 1807-1813 (1993))

HEK2 (Bohme et al. Oncogene 8, 2857-2862 (1993))

Eek, Erk (Chan and Watt, Oncogene 6, 1057-1061 (1991))

Ehk1, Ehk2 (Maisonpierre et al. Oncogene 8, 3277-3288 (1993))

Homologs for some of these receptors have been identified in otherspecies (Wicks et al. Proc. Natl. Acad. Sci. USA 89, 1611-1615 (1992));Gilardi-Hebenstreit et al. Oncogene 7, 2499-2506 (1992)). The expressionpatterns and developmental profiles of several family members suggestthat these receptors and their ligands are important for theproliferation, differentiation and maintenance of a variety of tissues(Nieto et al. Development 116, 1137-1150 (1992)). Structurally, EPHsub-family members are characterized by an Ig-like loop, a cysteine richregion, and two fibronectin-type repeats in their extracellular domains.The amino acid sequences of the catalytic domains are more closelyrelated to the SRC sub-family of cytoplasmic PTKs than to any of thereceptor PTKs. Among the catalytic domains of receptor PTKs, the EPHsub-family is most similar in amino acid sequence to the epidermalgrowth factor receptor sub-family.

It is an object of the invention to identify novel receptors belongingto the EPH sub-family. A directed PCR approach has been used to identifyfive human EPH-like receptors from a human fetal brain cDNA library.These receptors are designated HEK4, HEK5, HEK7, HEK8, and HEK11. Therelationship of these receptors to previously identified EPH-likereceptors is as follows:

HEK4 is the human homolog of Cek4 (chicken) and Mek4 (mouse) and isidentical to HEK (Boyd et al. J. Biol. Chem. 267, 3262-3267 (1992);Wicks et al., 1992) which was previously isolated from a human lymphoidtumor cell line.

HEK5 is the human homolog of Cek5, a full-length eph-like receptor clonefrom chicken. A portion of the HEK5 sequence was previously disclosed asERK, a human clone encoding about sixty amino acids (Chan and Watt,1991)

HEK7 is the human homolog of Cek7 isolated from chicken.

HEK8 is the human homolog of Cek8 a full-length clone from chicken andSek, a full-length clone from mouse. (Nieto et al., 1992; Sajjadi etal., 1991)

HEK11 does not have a known non-human homolog. With the addition of thenew members HEK5, HEK7, HEK8 and HEK11 and the report of a PCR fragmentencoding an eph-like receptor (Lai & Lemke Neuron 6, 691-704 (1991)), atotal of twelve distinct sequences that represent EPH-like receptorshave been published, making it the largest known sub-family of PTKs.

It is a further object of the invention to generate soluble EPH-likereceptors and antibodies to EPH-like receptors. Soluble receptors andantibodies are useful for modulating EPH-like receptor activation.

SUMMARY OF THE INVENTION

The present invention provides novel EPH-like receptor protein tyrosinekinases. More particularly, the invention provides isolated nucleicacids encoding four novel members of the sub-family of EPH-like receptorPTKs which are referred to collectively as HEKs (human-eph likekinases). Also encompassed are nucleic acids which hybridize understringent conditions to EPH-like receptor nucleic acids. Expressionvectors and host cells for the production of receptor polypeptides andmethods of producing receptors are also provided.

Isolated polypeptides having amino acid sequences of EPH-like receptorsare also provided, as are fragments and analogs thereof. Antibodiesspecifically binding the polypeptides of the invention are included.Also comprehended by the invention are methods of modulating theendogenous activity of an EPH-like receptor and methods for identifyingreceptor ligands.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the nucleotide and predicted amino acid sequence of theHEK5 receptor.

FIG. 2 shows the nucleotide and predicted amino acid sequence of theHEK7 receptor.

FIG. 3 shows the nucleotide and predicted amino acid sequence of theHEK8 receptor.

FIG. 4 shows the nucleotide and predicted amino acid sequence of theHEK11 receptor.

FIG. 5 shows the comparison of the amino acid sequences of the human EPHreceptor sub-family. The multiple sequence alignment was done using theLineUp program included in the Genetics Computer Group sequence analysissoftware package (Genetics Computer Group, (1991), Program Manual forthe GCG Package, Version 7, April 1991, Madison, Wis., USA 53711). Dotsindicate spaces introduced in order to optimize alignment. The predictedtransmembrane domains and signal sequences of each receptor areindicated by underlining and italics, respectively. Cysteine residuesconserved throughout the sub-family are indicated with asterisks. Arrowsindicate the tyrosine kinase catalytic domain. Amino acid sequences ofEPH, ECK and HEK2 were taken from the appropriate literature references.

FIG. 6 shows the molecular phylogeny of the EPH sub-family of receptorprotein tyrosine kinases. Catalytic domain sequences were analyzed asdescribed by Hanks and Quinn, 1991. The scale bar represents anarbitrary evolutionary difference unit. The EPH branch, which has beenshown with a discontinuity for the sake of compactness, is 23.5 units inlength.

FIGS. 7-11 show Northern blot analyses of the tissue distribution of theHEK receptors. Receptor cDNA probes, labeled with ³²P, were hybridizedto either 2 μg of poly A⁺ RNA from human tissues (panel A, Clontech) or10 μg of total RNA from rat tissues (panel B). Sizes of the transcriptswere determined by comparison with RNA molecular weight markers(Bethesda Research Labs, Gaithersburg, Md.). FIG. 7, HEK4; FIG. 8, HEK5;FIG. 9, HEK7; FIG. 10; HEK8; FIG. 11; HEK 11.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to novel EPH-like receptor proteintyrosine kinases. More particularly, the invention relates to isolatednucleic acids encoding four novel members of the sub-family of EPH-likereceptor PTKs. These four members are designated herein as HEK (humaneph-like kinases). Nucleic acids encoding HEK receptors were identifiedin a human fetal brain cDNA library using oligonucleotide probes toconserved regions of receptor PTKs and EPH-like receptor PTKs. Thepredicted amino acid sequences of three HEK receptors had extensivehomology in the catalytic domain to previously identified EPH-likereceptors Cek5, Cek7 and Cek8 isolated from chicken and, accordingly,are designated HEK5, HEK7 and HEK8. The predicted amino acid sequence ofthe fourth HEK receptor revealed that it was not a homolog of anypreviously identified EPH-like receptor. It is designated HEK11. It isunderstood that the term “HEKs” comprises HEK5, HEK7, HEK8 and HEK11 aswell as analogs, variants, and mutants thereof which fall within thescope of the invention.

The invention encompasses isolated nucleic acids selected from the groupconsisting of:

(a) the nucleic acids set forth in any of SEQ ID NO: 10, SEQ ID NO: 12,SEQ ID NO: 14, or SEQ ID NO: 16 and their complementary strands;

(b) a nucleic acid hybridizing to the coding regions of the nucleicacids in any of SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ IDNO: 16 under stringent conditions; and

(c) a nucleic acid of (b) which, but for the degeneracy of the geneticcode, would hybridize to the coding regions of the nucleic acids in anyof SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16. Thenucleic acids of the invention preferably hybridize to HEK5, HEK7, HEK8,or HEK11 coding regions under conditions allowing up to about 5%nucleotide mismatch based upon observed nucleic acid identities amongknown human or nonhuman EPH-like receptors. An example of such acondition is hybridization at 600 in 1M Na⁺ followed by washing at 60°in 0.2×SSC. Other hybridization conditions may be ascertained by oneskilled in the art which allow base pairing with similar levels ofmismatch.

In a preferred embodiment, the isolated nucleic acids encodepolypeptides having the amino acid sequences of HEK5, HEK7, HEK8 orHEK11. A nucleic acid includes cDNA, genomic DNA, synthetic DNA or RNA.Nucleic acids of this invention may encode full-length receptorpolypeptides having an extracellular ligand-binding domain, atransmembrane domain, and a cytoplasmic domain, or may encode fragmentssuch as extracellular domains which are produced in a soluble, secretedform. Nucleic acid constructs which produce soluble HEK receptors aredescribed in Example 3. Polypeptides and fragments encoded by thenucleic acids have at least one of the biological activities of anEPH-like receptor protein tyrosine kinase, such as the ability to bindligand.

The invention also encompasses nucleic acids encoding chimeric proteinswherein said proteins comprise part of the amino acid sequence of a HEKreceptor linked to an amino acid sequence from a heterologous protein.One example of such a chimeric protein is an extracellular domain of aHEK receptor fused to a heterologous receptor cytoplasmic domain.Example 5 describes the construction and expression of a chimericreceptor comprising the HEK8 extracellular domain with the trkBcytoplasmic domain and a second chimeric receptor comprising the HEK11extracellular domain with the trkB cytoplasmic domain. HEK receptors mayalso be fused to other functional protein domains, such as an Ig domainwhich acts as an antibody recognition site.

The nucleic acids of the present invention may be linked to heterologousnucleic acids which provide expression of receptor PTKs. Suchheterologous nucleic acids include biologically functional plasmids orviral vectors which provide genetic elements for transcription,translation, amplification, secretion, etc. One example of an expressionvector suitable for producing EPH-like receptors of the presentinvention is pDSRα which is described in Example 3. It is understoodthat other vectors are also suitable for expression of EPH-likereceptors in mammalian, yeast, insect or bacterial cells. In addition,in vivo expression of nucleic acids encoding EPH-like receptor PTKs isalso encompassed. For example, tissue-specific expression of EPH-likereceptors in transgenic animals may be readily effected using vectorswhich are functional in selected tissues.

Host cells for the expression of EPH-like receptor PTKs will preferablybe established mammalian cell lines, such as Chinese Hamster Ovary (CHO)cells or NIH 3T3 cells, although other cell lines suitable forexpression of mammalian genes are readily available and may also beused. Such host cells are transformed or transfected with nucleic acidconstructs suitable for expression of an EPH-like receptor. Transformedor transfected host cells may be used to produce suitable quantities ofreceptor for diagnostic or therapeutic uses and to effect targetedexpression of EPH-like receptors in selected adult tissues, such asbrain, kidney, and liver, or in embryonic or rapidly dividing tissues.

The present invention provides purified and isolated polypeptides havingat least one of the biological properties of an EPH-like receptor (e.g.ligand binding, signal transduction). The isolated polypeptides willpreferably have an amino acid sequence as shown in any of SEQ ID NO: 10,SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO: 16. Polypeptides of thisinvention may be full-length polypeptides having an extracellulardomain, a transmembrane domain, and a cytoplasmic domain, or may befragments thereof, e.g., those having only an extracellular domain or aportion thereof. It will be understood that the receptor polypeptidesmay also be analogs or naturally-occurring variants of the amino acidsequences shown in SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 or SEQ IDNO: 16. Such analogs are generated by amino acid substitutions,deletions and/or insertions using methods available in the art.

Polypeptides of the invention are preferably the product of expressionof an exogenous DNA sequences, i.e., EPH-like receptors are preferablyproduced by recombinant means. Methods of producing EPH-like receptorscomprising culturing host cells which have been transformed ortransfected with vectors expressing an EPH-like receptor are alsoencompassed. EPH-like receptors, particularly fragments, may also beproduced by chemical synthesis. The polypeptides so produced may beglycosylated or nonglycosylated depending upon the host cell employed,or may have a methionine residue at the amino terminal end. Thepolypeptides so produced are identified and recovered from cell culturesemploying methods which are conventional in the art.

EPH-like receptors of the present invention are used for the productionof antibodies to the receptors. Antibodies to HEK receptors have beendescribed in Example 4. Antibodies which recognize the polypeptides ofthe invention may be polyclonal or monoclonal and may be bindingfragments or chimeric antibodies. Such antibodies are useful in thedetection of EPH-like receptors in diagnostic assays in the purificationof receptor, and in the modulation of EPH-like receptor activation.

As described in co-pending and co-owned U.S. Ser. No. 08/145,616, theonly known ligand for an EPH-like receptor is a protein which binds toand induces phosphorylation of the eck receptor. The ECK receptor ligandwas previously identified as B61. (Holzman et al. Mol. Cell. Biol. 10,5830-5838 (1990)). The availability of ECK receptor was important forthe identification of a ligand since B61, although known, had not beenpreviously implicated as an ECK receptor ligand. Therefore, EPH-likereceptors having ligand binding domains are useful for theidentification and purification of ligands. Polypeptides of the presentinvention may be used to identify and purify ligands for HEK5, HEK7,HEK8 and HEK11 receptors. Binding assays for the detection of potentialligands may be carried out in solution or by receptor immobilization ona solid support using-methods such as those described in co-pending andco-owned U.S. Ser. No. 08/145,616. Such assays may employ an isolatedligand binding domain of a HEK receptor. Alternatively, a HEK ligandbinding domain fused to an Ig domain may be used to detect the presenceof HEK ligand on cell surfaces.

Soluble EPH-like receptors may be used to modulate (i.e., increase ordecrease) the activation of the cell-associated receptors, typically bycompeting with the receptor for unbound ligand. Modulation of EPH-likereceptor activation may in turn alter the proliferation and/ordifferentiation of receptor-bearing cells. For example, based upon theobserved tissue distribution of the receptors of this invention (seeTable 5), soluble HEK7 receptor is likely to primarily affectproliferation and/or differentiation of brain cells, while soluble HEK5receptor may affect primarily brain and pancreatic cells, althougheffects of HEK5 receptor on other tissues may not be excluded.

Antibodies to EPH-like receptors are useful reagents for the detectionof receptors in different cell types using immunoassays conventional tothe art. Antibodies are also useful therapeutic agents for modulatingreceptor activation. Antibodies may bind to the receptor so as todirectly or indirectly block ligand binding and thereby act as anantagonist of receptor activation. Alternatively, antibodies may act asan agonist by binding to receptor so as to faciliate ligand binding andbring about receptor activation at lower ligand concentrations. Inaddition, antibodies of the present invention may themselves act as aligands by inducing receptor activation. It is also contemplated thatantibodies to EPH-like receptors are useful for selection of cellpopulations enriched for EPH-like receptor bearing cells. Suchpopulations may be useful in cellular therapy regimens where it isnecessary to treat patients which are depleted for certain cell types.

The isolated nucleic acids of the present inventions may be used inhybridization assays for the detection and quantitation of DNA and/orRNA coding for HEK5, HEK7, HEK8, HEK11 and related receptors. Suchassays are important in determining the potential of various cell typesto express these receptors and in determining actual expression levelsof HEK receptors. In addition, the nucleic acids are useful fordetecting abnormalities in HEK receptor genes, such as translocations,rearrangements, duplications, etc.

Therapeutic regimens involving EPH-like receptors will typically involveuse of the soluble form of the receptor contained in a pharmaceuticalcomposition. Such pharmaecutical compositions may containpharmaceutically acceptable carrier, diluents, fillers, salts, buffers,stabilizers and/or other materials well known in the art. Furtherexamples of such constituents are described in Remington'sPharmaceutical Sciences 18th ed., A. R. Gennaro, ed. (1990).Administration of soluble EPH-like receptor compositions may be by avariety of routes depending upon the condition being treated, althoughtypically administration will occur by intravenous or subcutaneousmethods. Pharmaceutical compositions containing antibodies to EPH-likereceptors will preferably include mouse-human chimeric antibodies orCDR-grafted antibodies in order to minimize the potential for an immuneresponse by the patient to antibodies raised in mice. Other componentsof anti-EPH antibody compositions will be similar to those described forsoluble receptor.

The amount of soluble Eph-like receptors or anti-Eph antibody in apharmaceutical composition will depend upon the nature and severity ofthe condition being treated. Said amount may be determined for a givenpatient by one skilled in the art. It is contemplated that thepharmaceutical compositions of the present invention will contain about0.01 μg to about 100 mg of soluble receptor or anti-Eph antibody per kgbody weight.

A method for modulating the activation of an EPH-like receptor PTK isalso provided by the invention. In practicing this method, atherapeutically effective amount of a soluble EPH-like receptor or ananti-EPH antibody is administered. The term “therapeutically effectiveamount” is that amount which effects an increase or decrease in theactivation of an EPH-like receptor and will range from about 0.01 μg toabout 100 mg of soluble receptor or anti-EPH antibody per kg bodyweight. In general, therapy will be appropriate for a patient having acondition treatable by soluble receptor or anti-EPH antibody and it iscontemplated that such a condition will in part be related to the stateof proliferation and/or differentiation of receptor-bearing cells. Basedupon the tissue distribution of HEK receptors shown in Table 4,treatment with the pharmaceutical compositions of the invention may beparticularly indicated for disorders involving brain, heart, muscle,lung, or pancreas. However, some HEK receptors are displayed on a widevariety of tissues, so it is understood that the effects of modulatingreceptor activation may not be limited to those tissues describedherein.

The following examples are offered to more fully illustrate theinvention, but are not to be construed as limiting the scope thereof.Recombinant DNA methods used in the following examples are generally asdescribed in Sambrook et al. Molecular Cloning: A Laboratory Manual ColdSpring Harbor Laboratory Press, 2nd ed. (1989)

EXAMPLE 1

Cloning and Sequencing of HEK Receptor cDNA

We have isolated clones for five members of the EPH sub-family ofreceptor PTKs from a human fetal brain cDNA library. Oligonucleotideswere designed based on conserved amino acid sequences within the kinasedomain. Primer I was based on the amino acid sequenceTrp-Thr-Ala-Pro-Glu-Ala-Ile (SEQ ID NO: 1), which is well-conservedamong PTKs of many families. Primer II was based on the sequenceVal-Cys-Lys-Val-Ser-Asp-Phe-Gly (SEQ ID NO: 2), which is invariant amongEPH sub-family members but, except for the sequence Asp-Phe-Gly, israrely found in other PTKs. Fully degenerate oligonucleotidescorresponding to reverse translations of these protein sequences weresynthesized and utilized as primers in a polymerase chain reaction (PCR)with disrupted phage from a human fetal brain cDNA library as thetemplate. The products of this PCR reaction were cloned into the plasmidvector pUC19 and the nucleotide sequence of the inserts was determined.Of the 35 PCR inserts sequenced, 27 were recognizable as portions of PTKgenes. Their correspondence to previously published sequences issummarized in Table 1. TABLE 1 Receptor PCR Products Number of ClonesElk VCKVSDFGLSRYLQDDTSDPTYTSSLGGKIPVRWTAPEAI (SEQ ID NO: 3) 2 HEK4, HEK7VCKVSDFGLSRVLEDDPEAAYTT RGGKIPIRWTAPEAI (SEQ ID NO: 4)  5* HEK5VCKVSDFGLSRFLEDDTSDPTYTSALGGKIPIRWTAPEAI (SEQ ID NO: 5) 8 HEK8VCKVSDFGMSRVLEDDPEAAYTT RGGKIPIRWTAPEAI (SEQ ID NO: 6) 4 HEK11VCKVSDFGLSRVIEDDPEAVYTTT GGKIPVRWTAPEAI (SEQ ID NO: 7) 1 SRC VCKVSDFGLARLIEDNEYTARQ GAKFPIKWTAPEAI (SEQ ID NO: 8)  6* PDGF-βVCKVSDFGLARDIMRDSNYISK GSTFLPLKWTAPEAI (SEQ ID NO: 9) 1An asterisk indicates that different nucleic acid sequences encoded theamino acid sequence shown.

Six PCR inserts predict amino acid sequences which are identical to aportion of SRC, although they comprise two distinct nucleotidesequences. One insert appears to code for the human platelet derivedgrowth factor (PDGF)-β receptor. The remaining 18 PCR inserts consist of6 distinct nucleotide sequences, all of which appear to be fragments ofEPH sub-family members. One of the sequence predicts an amino acidsequence identical to the corresponding region of rat Elk (Lhotak etal., 1991)) and is likely to represent its human homolog. Two insertspredict amino acid sequences which match the translation of the PCRfragment tyro-4 (Lai and Lemke, 1991)) but are clearly distinct at thenucleotide level while two others correspond to tyro-1 and tyro-5. Thesixth PCR insert has a previously unreported EPH-related sequence. Sincefive of the clones contained portions of potential EPH sub-familymembers for which full-length sequences had not been reported, each wasradiolabeled and used as a probe to screen a human fetal brain cDNAlibrary. Several clones corresponding to each of the five probes wereisolated. For each of the five receptors, the nucleotide sequence of theclone containing the largest portion of the predicted coding region wasdetermined.

A single cDNA clone containing the complete coding region was isolatedonly for HEK4. The portions of HEK5, HEK7, HEK10 and HEK11 coding forthe amino terminus of these receptors were not found in any of theclones. In order to obtain the complete coding sequence, the RapidAmplification of cDNA Ends (RACE) technique was employed. In some cases,more than one round of RACE was necessary to obtain the missing portionof the coding region. Using this strategy, complete coding sequenceswere obtained for all clones except HEK7 which lacked the completeleader sequence. The DNA sequences of HEK5, HEK7, HEK8 and HEK11 areshown in FIGS. 1-4, respectively, and in SEQ ID NO: 10 (HEK5), SEQ IDNO: 12 (HEK7), SEQ ID NO: 14 (HEK 8) and SEQ ID NO: 16 (HEK11). Theamino acid sequences are shown in SEQ ID NO: 11 (HEK5), SEQ ID NO: 13(HEK7), SEQ ID NO: 15 (HEK8) and SEQ ID NO: 17 (HEK 11).

EXAMPLE 2

Analysis of HEK Receptor Sequences

HEK5, HEK7, HEK8 and HEK11 represent novel human EPH sub-family members,although homologs for all except HEK11 have been isolated from otherspecies. We refer to human EPH receptor sub-family members as HEKs(human EPH-like kinases) following the nomenclature of Wicks et al.,1992). We have chosen names and numbers for these receptors tocorrespond with previously discovered members of the family in chicken(Ceks) and in mouse (Mek) (Sajjadi et al. 1991; Sajjadi and Pasquale,1993; Pasquale, 1991). Extending the convention of designating thespecies of origin by the first letter, we refer to the rat homologs ofthe HEK receptors as Reks (rat EPH-like kinases).

HEK4 is the human homolog of the chicken receptor Cek4 (91% amino acididentity in the catalytic domain) and the mouse receptor Mek4 (96% aminoacid identity in the catalytic domain). The amino acid sequence of HEK5is very closely related (96% amino acid identity in the catalyticdomain) to the chicken receptor Cek5 (Pasquale et al. J. Neuroscience12, 3956-3967 (1992); Pasquale, 1991). HEK7 is probably the humanhomolog of the recently reported Cek7 (Sajjadi and Pasquale, 1993). HEK8is likewise very closely related to Sek (Gilardi-Hebenstreit et al.,1992)) and Cek8 (95% amino acid identity in the catalytic domain)(Sajjadi and Pasquale, 1993)). The human homologs for Cek6 and Cek9 haveyet to be reported, while the human homolog of Cek10 has just recentlybeen published. One of our human receptors has no close relatives inother species and apparently represents a novel member of the EPHsub-family. We have designated this receptor HEK11, assuming that humanhomologs for Cek 9 and 10 will be named HEK9 and HEK10, respectively. Asummary of known EPH sub-family members is shown in Table 2. TABLE 2 EPHreceptor sub-family members Human Non-human homologs EPH None identifiedECK None identified None identified^(#) Eek HEK4* Cek4, Mek4 HEK5 Cek5,Nuk, ERK None identified^(#) Cek6, Elk HEK7 Cek7, Ehk1 HEK8 Cek8, SekNone identified^(#) Cek9 HEK2 Cek10 HEK11 None identified Noneidentified Ehk2*published by Wicks et.al., 1992 as HEK^(#)Using the present nomenclature, the predicted human homolog of Eekis designated HEK3. For Cek6, the predicted human homolog is designatedHEK6; For Cek9, the predicted human homolog is designated HEK9.

The predicted amino acid sequences of the four novel receptor clones andthe previously known EPH sub-family members ECK (SEQ ID NO: 18), EPH(SEQ ID NO: 19), HEK2 (SEQ ID NO: 20) and HEK4 (SEQ ID NO: 21) werealigned as shown in FIG. 5. The four clones are closely related to eachother and to the known EPH sub-family members. The extracellular domainsequences of all four novel receptors contain the Ig-loop,fibronectin-type III repeats, and cysteine-rich region characteristic ofEPH sub-family members. The positions of the 20 cysteine residues areconserved among all sub-family members. Also completely conserved is theportion of the catalytic domain used as the basis for the EPH sub-familyspecific primer (Val-Cys-Lys-Val-Ser-Asp-Phe-Gly, SEQ ID NO: 2, aminoacids 757-764 in FIG. 5). Table 3 summarizes the percentage of sequenceidentity between pairs of human EPH sub-family members. The lowerportion of the table shows percent amino acid identity in the catalyticdomain while the upper half shows percent amino acid identity in theextracellular region. The amino acid sequences of the EPH-like receptorsare extremely well-conserved (60-89% amino acid identity) in thecatalytic region but not as highly conserved in the extracellular region(38-65% amino acid identity), as would be expected for members of thesame receptor sub-family. TABLE 3 Eph family amino acid sequencecomparison extracellular domains EPH ECK HEK4 HEK5 HEK7 HEK8 HEK2 HEK11EPH * 47 42 38 40 43 40 42 ECK 62 * 47 41 45 46 41 46 HEK4 62 76 * 53 6561 51 59 HEK5 60 74 81 * 52 53 63 51 HEK7 61 76 89 83 * 62 48 61 HEK8 6276 86 85 88 * 52 57 HEK2 61 74 81 89 82 83 * 48 HEK11 60 74 83 83 85 8580 * Catalytic domainsNumbers shown are precent identity

Pairwise comparisons of amino acid sequences can be used to constructphylogenetic trees depicting the evolutionary relatedness of a family ofmolecules. FIG. 6 is such a tree, which summarizes the relationshipsamong the EPH sub-family members only one family member is shown fromeach group of cross-species homologs and the human representative wasused whenever possible (refer to Table 2 for a summary of cross-specieshomologs). The branch lengths represent the degree of divergence betweenmembers. It has been shown previously that the EPH sub-family lies on abranch evolutionarily closer to the cytoplasmic PTKs than to otherreceptor PTKs (Lindberg and Hunter, 1993). Interestingly, the furtherone moves up the tree, the more closely related the receptors become andexpression becomes more localized to the brain.

EXAMPLE 3

Construction and Expression of HEK Receptor Extracellular Domains

Soluble extracellular forms of HEK receptor proteins were constructed bydeletion of DNA sequences encoding transmembrane and cytoplasmic domainsof the receptors and introduction of a translation stop codon at the 3′end of the extracellular domain. A construct of the HEK5 extracellulardomain had a stop codon introduced after lysine at position 524 as shownin FIG. 1; the HEK7 extracellular domain was constructed with a stopcodon after glutamine at position 547 as shown in FIG. 2; the HEK 8extracellular domain was constructed with a stop codon after threonineat position 547 as shown in FIG. 3.

HEK extracellular domain was amplified from a human fetal brain cDNAlibrary by PCR using primers 5′ and 3′ to the extracellular domaincoding region.

For HEK5, the primers (SEQ ID NO: 22) 5′ CTGCTCGCCGCCGTGGAAGAAACG and(SEQ ID NO: 23) 5′ GCGTCTAGATTATCACTTCTCCTGGATGCTTGTCTGGTA

were used to amplify the extracellular domain and to provide arestriction site for cloning into plasmid pDSRα. In addition, thefollowing primers were used to provide a translational start site, theelk receptor signal peptide for expression; and a restriction site forcloning into pDSRα:5′ GCGGTCGACGCCGCCGCCATGGCCCTGGATTGCCTGCTGCTGTTCCTCCTG; (SEQ ID NO: 24)and 5′ CGTTTCTTCCACGGCGGCGAGCAGAGATGCCAGGAGGAACAGCAGCAGGCAATC (SEQ IDNO: 25)

The resulting construct resulted in fusion of DNA encoding the elksignal sequence Met-Ala-Leu-Asp-Cys-Leu-Leu-Leu-Phe-Leu-Leu-Ala-Ser (SEQID NO: 26) to the first codon of the HEK5 receptor.

The resulting HEK5 extracellular domain was cloned into pDSRα afterdigestion with SalI and XbaI and transfected into CHO cells forexpression.

HEK8 extracellular domain was amplified from a human fetal brain cDNAlibrary by PCR using primers 5′ and 3′ to the extracellular domaincoding region. For HEK8, the primers5′ GAATTCGTCGACCCGGCGAACCATGGCTGGGAT and5′ GAATTCTCTAGATTATCATGTGGAGTTAGCCCCATCTCwere used to amplify the extracellular domain and to provide restrictionsites for cloning into plasmid pDSRα.

The resulting HEK8 extracellular domain was cloned into pDSRα afterdigestion with SalI and XbaI and transferred CHO cells for expression.

HEK7 extracellular domain was amplified from a human fetal brain cDNAlibrary by PCR using primers 5′ and 3′ to the extracellular domaincoding region. For HEK7, the primers5′ TTCGCCCTATTTTCGTGTCTCTTCGGGATTTGCGACGCTCTCCGGAC CCTCCTGGCCAGC and5′ GAATTCTCTAGATTATCACTGGCTTTGATCGCTGGAT

were used to amplify the extracellular domain. In addition, thefollowing primers were used to provide a translational start site, theHEK8 receptor signal peptide sequence, and restriction site for cloninginto plasmid pDSRα. 5′ GAATTCGTCGACCCGGCGAACCATGGCTGGGATTTTCTATTTCGCCCTATTTTCGTGTCT 5′ GAATTCTCTAGATTATCACTGGCTTTGATCGCTGGAT

The resulting construct resulted in fusion of DNA incoding HEK8 signalsequenceMet-Ala-Gly-Ile-Phe-Tyr-Phe-Ala-Leu-Phe-Ser-Cys-Leu-Phe-Gly-Ile-Cys-Aspto the first codon of the HEK7 receptor.

The resulting HEK7 extracellular domain was cloned into pDSRα afterdigestion with SalI and XbaI and transfected into CHO cells forexpression.

EXAMPLE 4

Antibodies to HEK Receptors

Antibodies to HEK receptor proteins were generated which recognize theextracellular domain by using bacterial fusion proteins as the antigen.Antibodies were also generated which recognize the cytoplasmic domain byusing synthetic peptides as the antigen.

The methodology employed has been previously described (Harlow and Lane,In Antibodies: A Laboratory Manual, 1988). For the extracellular domainantibodies, cDNAs were inserted into the pATH vector (see Table 4 forthe regions of each receptor encoded by this construct). Theseconstructs were expressed in bacteria and the resultant TrpE-fusionproteins were purified by SDS-polyacrylamide gel electrophoresis. Forthe cytoplasmic domain anti-peptide antibodies, peptides weresynthesized (see Table 4 for the sequences) and covalently coupled tokeyhole limpet hemocyanin. The fusion proteins and coupled peptides wereused as antigens in rabbits and antisera were generated andcharacterized as described (Harlow and Lane, 1988). Anti-peptideantibodies were affinity purified by using a SulfoLink kit (Pierce,Rockford Ill.). TABLE 4 HEK Receptor Antigens Amino Acids in ReceptorPeptide Sequences Fusion Protein HEK4 CLETQSKNGPVPV 22-159 HEK5CRAQMNQIQSVEV 31-168 HEK7 CMKVQLVNGMVPL 335-545  HEK8 CMRTQMQQMHGRIMVPV27-188 HEK11 CQMLHLHGTGIQV 187-503 

EXAMPLE 5

HEK/TrkB Chimeric Receptors

1. Generation of pSJA1 encoding rat trkB cytoplasmic domain.

All of the chimeric receptors are composed of the extracellular domainand the transmembrane region of one of the HEK receptors and theintracellular portion of rat trkB. To simplify each individualconstruction, an intermediate or parental plasmid, called RtrkB/AflII(or pSJA1), was generated. First, without altering the coded peptidesequence, an AflII site (CTTAAG) was introduced into position 2021(cytosine at position 2021 (C2021) to guanine at position 2026 (G2026,CTCAAG) of the rat trkB cDNA (Middlemas, et al., Mol. Cell. Biol. 11,143-153 (1991)) by PCR aided mutagenesis. Briefly, PCR primers weresynthesized based on the rat trkB cDNA sequence. Primer I encompassedC2003 to G2034 of the cDNA. This primer contained two mutations, acytosine to thymine (T) substitution at position 2023 (C2023T) and aninsertion of an adenine (A) in between T2013 and G2014. These mutationscreated the AflII site at position C2021 and an additional XhoI siteflanking the AflII site. Primer II was in the reverse directionencompassing T2141 to A2165 of the cDNA which bore an ApaI site. The PCRfragment produced with these primers and the rat trkB cDNA template wasdigested with XhoI and ApaI enzymes and sub cloned into the XhoI andApaI sites of an expression vector, pcDNA3 (InVitroGen), to generatepSJA1-b. Following, pSJA1-b was linearized with ApaI and ligated with aBanII digested rat trkB cDNA fragment (G2151 to G4697) to reconstitute alarger fragment (C2021 to G4697) including the coding sequence of thewhole intracellular domain of the rat trkB protein (L442 to G790) and1571 residues (A3131 to G4697) of the 1627 nucleotide 3′-end non-codingregion of the cDNA.

2. Generation of HEK8/rat trkB (pSJA5) chimera.

HEK8/rat trkB chimera was generated with a similar strategy as mentionedabove. A SalI/BsaI cDNA fragment was first isolated from plasmidTK10/FL13. This fragment included the nucleotide sequence from thebeginning to T1689 of the HEK8 cDNA (FIG. 3). Then, a pair ofoligonucleotides was synthesized based on the HEK8 cDNA sequence. Thesequence of the first oligonucleotide was the same as G1690 to C1740 ofthe Hek8 cDNA, with an additional C residue-added to its 3′-end. Thesecond oligonucleotide was in the reverse orientation of the HEK8 cDNA.It contained C1694 to C1740 of the HEK8 cDNA sequence and an additionalfive residue motif, TTAAG, at its 5′-end. These two oligonucleotideswere kinased and annealed with equal molar ratio, to create a doublestrand DNA fragment with the sequence of G1690 to C1740 of the HEK8 cDNAand with the BsaI and the AflII cohesive ends at its 5′ and 3′ ends,respectively. This fragment was ligated together with the SalI/BsaI cDNAfragment into XhoI/AflII linearized pSJA1 to generate the HEK8/RtrkB(pSJA5) chimerical construct.

3. Generation of HEK11/rat trkB (pSJA6) chimera.

To generate the HEK11/rat trkB chimera, a SalI/AccI fragment coveringthe sequence of nucleotide C1 to T1674 of the HEK11 cDNA (FIG. 4) wasfirst isolated from plasmid TK19T3. Then, a pair of oligonucleotides wassynthesized based on the HEK11 cDNA sequence. The first oligonucleotidehad the same sequence as from nucleotide A1666 to T1691 of the HEK11cDNA, which contained the AccI site. The second oligonucleotide was inthe reverse orientation of the HEK11 cDNA. It encompassed G1895 to T1919of the HEK11 cDNA sequence. An additional ten residue motif, CCCGCTTAAG,was added to the 5′-end of this oligonucleotide to introduce an AflIIsite, which would be used to link the external domain and thetransmembrane region of the HEK11 receptor to the intracellular domainof the rat trkB cDNA cloned in pSJA1 in the same reading frame. PCR wasperformed with these oligonucleotides as primers and the HEK11 cDNA astemplate. The PCR fragment was digested with AccI and AflII enzymes andligated with the SalI/AccI cDNA fragment and the XhoI/AflII linearizedpSJA1 to generate the HEK11/rat trkB (pSJA6) chimerical construct.

EXAMPLE 6

Tissue Distribution of HEK Receptors

The distribution of mRNA expression for HEK4, HEK5, HEK7, HEK8 and HEK11receptors in human and rat tissues was examined by Northern blothybridization.

Rat total RNA was prepared from tissues using the method of Chomczynskiand Sacchi (Anal. Biochem 162, 156-159 (1987)). The RNA was separated byformaldehyde-agarose electrophoresis and transferred to Hybond-Nmembranes (Amersham, Arlington Heights, Ill.) using 20×SSC (Maniatis etal. 1982). The membrane was dried at 80° C. in vacuo for 30 minutes,then crosslinked for 3 minutes on a UV transilluminator (Fotodyne, NewBerlin, Wis.). The membrane was prehybridized for 2 hours at 42° C. in50% formamide, 5×SSPE, 5× Denhardt's, 0.2% SDS, and 100 μg/ml denaturedherring sperm DNA (Maniatis et al. 1982). Northern blots of human tissuewere purchased from Clontech (Palo Alto, Calif.). Probes were preparedby labeling the fragment of cDNA which encoded the extracellular domainof the receptor with ³²P-dCTP using a hexanucleotide random priming kit(Boehringer Mannheim, Indianapolis, Ind.) to a specific activity of atleast 1×10⁹ cpm/ug. The probe was hybridized to the membrane at aconcentration of 1-5 ng/ml at 42° C. for 24 to 36 hours in a buffersimilar to the prehybridization buffer except that 1× Denhardt's wasused. After hybridization, the membranes were washed 2 times for 5minutes each in 2×SSC, 0.1% SDS at room temperature followed by two 15minute washes in 0.5×SSC, 0.1% SDS at 55° C. Blots were exposed for 1-2weeks using Kodak XAR film (Kodak, Rochester, N.Y.) with a DupontLightning Plus intensifying screen. The results are shown in FIGS. 7-11.

Homologs for HEK4 have been previously identified from mouse, chicken,and rat. In the adult mouse, expression is detected primarily in thebrain and testis (Sajjadi et al. 1991). A slightly different pattern wasfound in adult chicken tissues, with the main sources of expressionbeing the brain, liver, and kidney. Lower levels of expression weredetectable in the lung and heart (Marcelle & Eichmann, Oncogene 7,2479-2487 (1992)). A fragment of the Rek4 gene (tyro-4) has beenisolated and used to look at tissue expression in the adult rat (Sajjadiet al. 1991). The brain was the only tissue that expressed Rek4 mRNA.However, RNA from lung or testis were not examined. Previous studies onHEK4 only looked at the expression of the mRNA in cell lines, where itwas found in one pre-B cell line and two T-cell lines (Wicks et al.1992). The significance of this with regard to in vivo expressionremains to be determined. In this study we have looked at the HEK4expression in human tissues, and also the expression of Rek4 in rattissues. The HEK4 mRNA corresponds to a single transcript with a size ofabout 7 kb (FIG. 7A). HEK4 mRNA was most abundantly expressed inplacenta, with lower levels present in heart, brain, lung, and liver. Onprolonged exposures, trace amounts of mRNA were detectable in kidney andpancreas. Expression in the rat was more similar to that detected in themouse and chicken. Rek4 was expressed at the lowest levels of any of thefamily members characterized herein. A transcript of about 7 kb wasdetectable in rat lung, with a lower amount detectable in brain (FIG.7B). Also, a 4 kb transcript was expressed in rat testis. Because thetranscripts were barely detectable using total RNA, some of the otherrat tissues may contain amounts of Rek4 below the level of detection.

The expression of HEK5 in adult tissues has been previously studied inchicken and rat. Studies in the chicken have identified the Cek5 proteinin the brain and liver, with a smaller protein detected in theintestine. In the rat, the tyro-5 fragment detected mRNA expression onlyin the adult brain, though intestine was not examined (Lai and Lemke,1991). Our results show that HEK5 mRNA was expressed at much higherlevels than HEK4 and was found as transcripts of several sizes. The mostabundant mRNAs were of approximately 4.0 and 4.4 kb, with lesser amountsof higher molecular weight transcripts of 9.5 kb and longer (FIG. 8A).The HEK5 mRNA was most abundantly expressed in placenta, but was alsohighly expressed in brain, pancreas, kidney, muscle, and lung. Longerexposures of the blots revealed the presence of transcripts in heart andliver as well. The rat homolog of HEK5 (Rek5) showed a somewhat similarpattern of expression. Rek5 was most abundant in intestine, followed bybrain, kidney, lung, thymus, stomach, and ovary (FIG. 8B). Expressionwas not detectable in testis, muscle, heart, or liver. During ouranalysis of this family, we concluded that the rat Erk fragment (Chan &Watt, 1991) likely encodes a portion of the Rek5 receptor. Erkexpression was examined in several rat tissues and found only in thelung. The reason for the discrepancy between that report and what we andothers (Lai & Lemke, 1991) have found is unclear.

Homologs for HEK8 have been identified from chicken, mouse, and rat. Inthe adult chicken, a single Cek8 transcript was found to be expressed athigh levels in the brain, with expression also detected in the kidney,lung, muscle, and thymus. The expression of the mouse homolog of HEK8,Sek, has been detected as a single transcript with abundant expressionin the adult brain and lower expression in the heart, lung and kidney. Afragment of Rek8 (tyro-1) was used to look at expression in rat tissues,with expression found only in the brain (Lai & Lemke, 1991). We foundthat HEK8 mRNA was expressed at levels comparable to that of HEK5.Multiple transcripts were also observed, the most abundant at 7 kb and 5kb. The highest level of mRNA expression was seen in the brain, althoughsubstantial levels were detected in other tissues including heart, lung,muscle, kidney, placenta, and pancreas. Expression in liver was muchlower than in the other tissues. The only difference in expressionpatterns between human and mouse was expression in human muscle, alsoseen for Cek8 in chicken. Among the rat tissues, Rek8 was most highlyexpressed in the brain, followed by the lung, heart, and testis (FIG.10B). In contrast to HEK8, expression of Rek8 appeared to be lower inmuscle and kidney, two tissues where HEK8 was readily detectable. Inaddition, Rek8 was not expressed as a 5.0 kb transcript, as it was notvisible even on prolonged exposures.

During the analysis of this family, we deduced that HEK7 is the humanhomolog of Cek7. The only expression seen in adult chicken was an 8.5 kbtranscript found in the brain (Sajjadi & Pasquale, 1993). Of the fiveEPH sub-family members described here, HEK7 was the most restricted inits expression pattern. Analysis of human mRNA revealed significantexpression only in the brain, with a much lower level detectable in theplacenta (FIG. 9A). Prolonged exposures did not reveal expression in anyother tissue examined. Two prominent transcripts were found in brain,the most highly expressed with a size of 6 kb and the other with alength of 9 kb. In the placenta, however, only the 9 kb transcript wasdetected. Rek7 mRNA was expressed in a pattern similar to HEK7. Thehighest level of expression was found in brain, with a much lower levelin ovary (FIG. 9B). The transcripts were of similar size as for HEK7,with the 6 kb transcript detected only in brain.

HEK11 was expressed as several transcripts, with major mRNAs of length7.5, 6.0 and 3.0 kb and minor transcripts of 4.4 and 2.4 kb (FIG. 11A).All five mRNAs were expressed at the highest levels in brain, followedby heart. Placenta, lung and kidney had significant amounts of four ofthe five transcripts, with lower expression seen in muscle. Pancreas hadbarely detectable amounts of HEK11 mRNA, while liver had no detectableHEK11 transcript. Rek11 had a similar pattern of expression, with fourtranscripts (10, 7.5, 3.5 and 3.0 kb) detected in brain (FIG. 11B).

The relative level of mRNA expression for each of the five receptors inall tissues studied is summarized in Table 5. TABLE 5 TissueDistribution of HEK Receptors HEK4 HEK5 HEK7 HEK8 HEK11 Human Brain ++++ ++ +++ ++ Heart + + bd ++ + Kidney + + bd + + Liver + + bd + bdLung + + bd ++ + Muscle + + bd ++ + Pancreas + ++ bd + bd Placenta ++++++ bd ++ + Rat Brain + ++ +++ +++ ++ Heart bd bd bd + bd Intestine bd+++ bd bd bd Kidney bd ++ bd bd bd Liver bd bd bd bd bd Lung + + bd ++bd Muscle bd bd bd bd bd Ovary bd + + bd bd Stomach bd + bd bd bdTestis + bd bd + bd Thymus bd + bd bd bdbd = below detection

The transcripts for HEKs 4, 5, 8, and 11 were rather widely distributedin human tissue while HEK7 was specific for brain. Expression patternsbetween rat and human tissue were roughly comparable given that the ratblots were less sensitive due to the use of total RNA rather thanpolyA⁺. As was found for the Cek mRNAs by Sajjadi and Pasquale (Sajjadi& Pasquale, 1993), often there were several different size transcriptsdetected for a single receptor. The size distribution of the transcriptsappears to be both tissue and species specific. Previous work has shownthat the smaller transcript of Mek4 encodes a potentially secretedreceptor (Sajjadi et al. 1991).

The following sections describe Materials and Methods used to carry outexperiments described in Example 1.

Isolation, Cloning and Sequencing of HEK Receptor cDNAs

Fragments containing a portion of the catalytic domain of EPH sub-familyreceptors were generated using a polymerase chain reaction (PCR) withdisrupted phage from a human fetal brain cDNA library as a template. A10 μl aliquot of the cDNA library (Stratagene, La Jolla, Calif.) wastreated at 70° C. for 5 minutes to disrupt the phage particles, thencooled on wet ice. The disrupted phage were added to 10 μl of 10×Taqpolymerase buffer, 8 ul of 2 mM each DNTP, 100 picomoles of each primer,and 1.5 μl of Tag polymerase (Promega, Madison, Wis.) in a total volumeof 100 μl. The reaction was run for 35 cycles, each consisting of 1minute at 96° C., 1 minute at 50° C., and 2 minutes at 72° C. A 5minute, 72° C. incubation was added at the end to ensure completeextension. The primers used were degenerate mixtures of oligonucleotidesbased on amino acid sequences which are highly conserved among EPHsub-family members. 5′AGGGAATTCCAYCGNGAYYTNGCNGC′; (SEQ ID NO: 27)5′AGGGGATCCRWARSWCCANACRTC′. (SEQ ID NO: 28)

The products of the PCR reaction were digested with EcoRI and BamHI andcloned into M13 mp19 (Messing, Methods Enzymol. (1983)) for sequenceanalysis. The five clones which were identified as fragments of EPHreceptor sub-family members were labeled with ³²P-dCTP by random primingand each was used to screen Genescreen nitrocellulose filters (NEN,Boston, Mass.) containing plaques from the human fetal brain cDNAlibrary. Phage stocks prepared from positively screening plaques wereplated and rescreened with the same probe in order to obtain singleclones. cDNA inserts were transferred into pBluescript using the in vivoexcision protocol supplied with the cDNA library (Stratagene, La Jolla,Calif.). Nucleotide sequences were determined using Taq DyeDeoxyTerminator Cycle Sequencing kits and an Applied Biosystems 373Aautomated DNA sequencer (Applied Biosystems, Foster City, Calif.).

5′ Race

The 5′ ends of the cDNAs were isolated using a 5′ RACE kit (GIBCO/BRL,Gaithersburg, Md.) following the manufacturer's instructions. Excessprimers were removed after first strand cDNA synthesis usingultrafree-MC cellulose filters (30,000 molecular weight cutoff,Millipore, Bedford, Mass.). Amplified PCR products were digested withthe appropriate restriction enzymes, separated by agarose gelelectrophoresis, and purified using a Geneclean kit (Bio101, La Jolla,Calif.). The purified PCR product was ligated into the plasmid vectorpUC19 (Yanisch-Perron et al. Gene 33, 103-119 (1985)) which had beendigested with appropriate restriction enzymes and the ligation mixturewas introduced into host bacteria by electroporation. Plasmid DNA wasprepared from the resulting colonies. Those clones with the largestinserts were selected for DNA sequencing.

While the present invention has been described in terms of preferredembodiments, it is understood that variations and modifications willoccur to those skilled in the art. Therefore, it is intended that theappended claims cover all such equivalent variations which come withinthe scope of the invention as claimed.

1-34. (canceled)
 35. An isolated EPH-like receptor protein tyrosinekinase polypeptide or fragment thereof encoded by a nucleic acidselected from: a) a nucleic acid comprising a nucleic acid selected fromSEQ ID NO: 10 and SEQ ID NO: 12; b) a nucleic acid comprising a portionof a nucleic acid selected from SEQ ID NO: 10 and SEQ ID NO: 12, whereinthe portion encodes at least one of an extracellular domain and acytoplasmic domain; c) a nucleic acid hybridizing at 60° C. in 1M Na⁺followed by washing at 60° C. in 0.2×SSC to the complement of the codingregion of a nucleic acid selected from SEQ ID NO: 10 and SEQ ID NO: 12;d) a nucleic acid comprising a portion of a nucleic acid hybridizing at60° C. in 1M Na⁺ followed by washing at 60° C. in 0.2×SSC to thecomplement of the coding region of a nucleic acid selected from SEQ IDNO: 10 and SEQ ID NO: 12, wherein the portion encodes at least one of anextracellular domain and a cytoplasmic domain; e) a nucleic acid that is95% identical to a nucleic acid selected from SEQ ID NO: 10 and SEQ IDNO: 12; f) a nucleic acid that is 95% identical to a portion of anucleic acid selected from SEQ ID NO: 10 and SEQ ID NO: 12, wherein theportion encodes at least one of an extracellular domain and acytoplasmic domain; and g) a nucleic acid comprising a sequence which isdegenerate to a nucleic acid of any one of (a)-(f).
 36. The isolatedEPH-like receptor protein tyrosine kinase polypeptide or fragmentthereof of claim 35, wherein the polypeptide or fragment thereofcomprises an amino acid sequence selected from the amino acid sequenceof SEQ ID NO: 11 and the amino acid sequence of SEQ ID NO:
 13. 37. Theisolated EPH-like receptor protein tyrosine kinase polypeptide orfragment thereof of claim 35, wherein the polypeptide or fragmentthereof comprises a portion of an amino acid sequence selected from theamino acid sequence of SEQ ID NO: 11 and the amino acid sequence of SEQID NO: 13, wherein the portion comprises at least one of anextracellular domain and a cytoplasmic domain.
 38. The isolated EPH-likereceptor protein tyrosine kinase polypeptide or fragment thereof ofclaim 37, wherein the portion comprises an extracellular domain.
 39. Theisolated EPH-like receptor protein tyrosine kinase polypeptide orfragment thereof of claim 37, wherein the polypeptide or fragmentthereof comprises an amino acid sequence selected from amino acids 6 to524 of SEQ ID NO: 11 and amino acids 1 to 547 of SEQ ID NO:
 13. 40. Theisolated EPH-like receptor protein tyrosine kinase polypeptide orfragment thereof of claim 38, further comprising a heterologous receptorcytoplasmic domain.
 41. The isolated EPH-like receptor protein tyrosinekinase polypeptide or fragment thereof of claim 39, further comprising aheterologous receptor cytoplasmic domain.
 42. The polypeptide of claim35, wherein the polypeptide is obtained by expression of a nucleic acidtransformed or transfected into a procaryotic or eucaryotic host cell.